Bioconversion of Waste Biomass to Bioethanol

Introduction

The concept of biorefinery is an outcome of advancements in biotechnology, chemical engineering, bioresource technology, process chemistry, genetic engineering, industrial engineering and other cross-disciplinary areas, all of which support research and development on the conversion of alternative resources to value-added fuels, commodity chemicals and industrially relevant products. The production of biofuels and biochemicals from various waste biomass has gained immense interest in research and applications in the past few decades as far as the bio-based economy and circular economy are concerned (Nanda et al. 2015b; Sarangi and Nanda 2020). Renewable organic wastes including lignocellulosic feedstocks (e.g. agricultural biomass, forestry residues and energy crops), microalgae and other biogenic wastes (e.g. municipal solid waste, food waste, waste cooking oil, cattle manure, sewage sludge and industrial effluents) have huge potentials to produce biofuels through biological and thermochemical conversion for supplementing the global energy requirements (Nanda et al. 2016a; Nanda et al. 2016b; Nanda et al. 2016c; Nanda et al. 2016e; Reddy et al. 2016; Gong et al. 2017a; Gong et al. 2017b; Nanda et al. 2017c; Nanda et al. 2018b; Nanda et al. 2019). The biofuels produced from the above-mentioned lignocellulosic biomass and biogenic wastes are termed as second-generation biofuels (Nanda et al. 2018a).

Bioethanol is one of the second-generation biofuels and base chemicals produced from the biological conversion of lignocellulosic biomass and organic wastes. Bioethanol can be blended with petrol (or gasoline) in various proportions such as ЕЮ. E85 and E95 containing 10%, 85% and 95% of ethanol, respectively. The blending of ethanol at lower proportions with gasoline is preferred for use in the existing vehicular engines since higher proportion necessitates significant mechanical modifications to the automobile engines (Sarangi and Nanda 2018). Moreover, ethanol has an oxygen content of 35% and is completely soluble in water at 25°C, which causes technical issues in higher blending ratios with gasoline. The calorific value of ethanol (C2H5OH) is 21.2 MJ/kg while that of gasoline is 32.5 MJ/kg (Nanda et al. 2017b). Similarly, a few other fuel properties such as research octane number, motor octane number and the air-fuel ratio of ethanol are 129, 102 and 9, respectively (Nanda et al. 2017b).

This chapter gives an overview' of bioethanol production from w'aste lignocellulosic biomass. The chapter describes the potential of waste biomass for bioethanol production. It also provides insights on microbial fermentation for bioethanol production along with the biomass pretreatment and bioprocess parameters. The chapter concludes with a note on technical challenges and future possibilities.

Potential of Lignocellulosic Biomass

When compared to fossil fuels, waste plant biomass is considered to be economical, abundant and renewable, making them one of the most prominent and trusted sources of renewable energy (Sarangi and Nanda 2019a; Sarangi and Nanda 2019b; Sarangi and Nanda 2019c). Bioethanol was traditionally produced from first- generation feedstocks mostly comprising food crops and grains (e.g., maize, potatoes, wheat, cassava and other starch-based crops). However, the massive diversion of these food crops to biorefineries for bioethanol production contributed to a shortage in the food supply, rising food prices, the competition to cultivable lands and the socio-environmental unrest relating to the “food versus fuel” controversy (Nanda et al. 2015b).

With the unpopularity of first-generation bioethanol, soon emerged the second- generation bioethanol produced from inedible plant residues that have no competition to food supply and arable lands. These inedible plant residues are mostly the lignocellulosic biomass. The accessibility of biomass resources and their potential to produce biofuel should be focused to fulfill the demand of bioethanol. Food security should not be compromised by any nation w'hile addressing domestic energy security. Hence, it becomes an important consideration to resolve the energy crisis along with the suitable valorization of agricultural and forestry refuse as well as organic wastes economically and sustainably.

Cellulose constitutes about 35-55 wt% of lignocellulosic biomass followed by hemicelluloses (20-40 w't%) and lignin (10-25 w't%) (Nanda et al. 2013). Trace presence of inorganic ingredients (i.e. mineral matter and ash), nitrogenous compounds, waxes and extractives (e.g. pectin, ester, ether, resins, tannins, terpenoids, chlorophyll and other polar and non-polar components) is also found in lignocellulosic biomass (Okolie et al. 2019). Hydrogen and covalent bonds link cellulose and hemicellulose firmly with lignin, which makes the lignocellulosic framework recalcitrant to chemical agents and enzymes. Straight chains of D-glucose subunits are found in cellulose connected with (3-(l,4)-glycosidic bonds. Cellulose occurs in both amorphous and crystalline nature in biomass (Nanda et al. 2016d). Hemicelluloses are matrix polysaccharides containing both hexose sugars (e.g. glucose, rhamnose, galactose and mannose) and pentose sugars (e.g. xylose and arabinose) as well as sugar acids (e.g. glucuronic acid and galacturonic acid) (Okolie et al. 2020). Lignin is a highly branched cross-linked aromatic phenylpropane polymer with hydrophobic properties (Fougere et al. 2016). It is synthesized from phenylpropanoid precursors that lead to the synthesis of these polyphenolic aromatic compounds. The basic building blocks of lignin are p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol (Rana et al. 2018).

Upstream and Downstream Technologies in Bioethanol Production

The production of second-generation ethanol is typically a four-stage process such as: (i) biomass pretreatment and hydrolysis, (ii) enzymatic saccharification, (iii) microbial fermentation and (iv) product separation (Nanda et al. 2014a; Sarangi et al. 2020). A suitable physicochemical, chemimechanical or hydrothermal pretreatment causes structural modifications to lignocellulosic biomass, thus reducing cellulose crystallinity, depolymerizing lignin and separating hemicellulose (Nanda et al. 2014b). A hydrolytic pretreatment of biomass enhances the efficiency of subsequent enzyme-mediated saccharification of fermentable sugars (pentose and hexose) recovered from cellulose and hemicellulose. Enzymatic saccharification enhances the near-complete release of monomeric sugars from the hydrolyzed carbohydrates and polysaccharides in the biomass for microbial fermentation.

Several cellulolytic enzymes (e.g. cellulases), hemicellulases, lignin-modifying and lignin-degrading enzymes are involved in enzymatic saccharification and hydrolysis of lignocellulosic biomass (Parakh et al. 2020). In the next step, suitable microorganisms (individual or consortium) are used to ferment the monomeric sugars recovered from biomass because of pretreatment and enzymatic saccharification to the desired alcohol and other organic compounds (e.g., ethanol, butanol, methanol, acetone, etc.) (Sarangi and Nanda 2019b). In the final downstream stage, the products are separated from the fermentation media and purified based on their chemical and solvent properties through a wide variety of technologies such as distillation, gas stripping, liquid-liquid separation, adsorption, perstraction, pervaporation, supercritical CO, fractionation, etc. (Nanda et al. 2017a). The typical upstream and downstream process involved in the production of bioethanol from lignocellulosic biomass is illustrated in Figure 3.1.

Typical upstream and downstream steps involved in lignocellulosic biomass conversion to bioethanol

FIGURE 3.1 Typical upstream and downstream steps involved in lignocellulosic biomass conversion to bioethanol

Enzymatic Saccharification

Enzymatic hydrolysis plays a key role in determining the operating costs involved in the production of second-generation bioethanol. An efficient enzymatic saccharification could lead to the maximum recovery of fermentable monomeric sugars from the biomass, thus leading to an improved fermentation for bioethanol production. The first-generation biomass, which predominantly contains starch, is relatively easier to hydrolyze and ferment for bioethanol production. In contrast, lignocellu- losic biomass contains highly robust and biochemically stable lignin, which creates hindrances in the access of enzymes and other pretreatment agents for denaturing the cellulose-hemicellulose-lignin matrix and releasing the sugars (Nanda et al. 2015a).

Cellulases (i.e. cellulose-degrading enzymes) along with hemicellulase (i.e. hemi- cellulose-degrading enzymes) are utilized for a complete breakdown of all polysaccharides in lignocellulosic biomass into sugar monomers. Cellulases are a pivotal and prominent constituent of the enzyme cocktail used for saccharification because of the higher degree of polymerization of cellulose and their crystallinity than that of hemicellulose (Sukharnikov et al. 2011). Van der Waals interactions along with hydrogen bonds prevailing in between the glucose monomers are responsible for rendering recalcitrance to cellulose fibers. Therefore, reducing cellulose crystallinity and increasing amorphous cellulose moieties are key considerations for enzymatic saccharification. With specific functionalities of degrading cellulose, the types of cellulase enzymes are endocellulases, exocellulases, cellobiases (or (3-glucosidases), oxidative cellulases and cellulose phosphorylases.

A variety of glycoside hydrolases are used in enzymatic saccharification of complex lignocelluloses. The family of glycoside hydrolases includes cellulases, hemicellulases, pectin-degrading enzymes and lignin-degrading enzymes. More than 130 glycoside hydrolases families have been explored for the conversion of complex carbohydrates into simpler sugars (Lombard et al. 2014), out of which 40 are cellulolytic enzymes with the ability to achieve high-efficiency cellulose hydrolysis with well-coordinated synergy for bioethanol production (Liu et al. 2018).

Cellulases, hemicellulases and (3-glucosidases are the main constituents of the cocktail that manifest the conversion of polysaccharides into pentose and hexose monomers. The second-generation bioethanol production can be made cost-effective by engineering the bioprocess to recycle the expensive enzyme cocktail, which significantly adds to the process expenditures. Strategies should also be focused on enhancing the efficiency of enzymes either by process engineering or by upstream modifications (e.g. efficient biomass pretreatment with maximum hydrolysis). Another strategy is genetic engineering, which involves high-performance mutagenesis and engineered energy crops. Second, maximum hydrolysis can be achieved if an enzyme cocktail can act best under specified process conditions while retaining its thermal stability and viability. Enzymes recovered from extremophilic microorganisms could be a possible solution (Miller and Blum 2010).

Thermophilic enzymes can make bioprocess technology cost-effective as they enhance the overall performance of the process by preventing contamination of hydrolysates. In such a scenario, saccharification can perform at temperatures relatively higher than that preferred by contaminating microorganisms. Under ambient conditions, soil microbial heterotrophs are supported by their efficiencies to undergo enzymatic hydrolysis of lignocellulosic materials through natural decomposition, which is also an important process for carbon cycling in terrestrial ecosystems (Nanda et al. 2016f).

 
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